Our research program has attempted to unravel the complex regulatory processes which contribute to the regulation of cellular protein levels. As an example of this general problem, our focus has been, and will continue to be, on the key enzyme in the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme-A reductase. Our Specific Aims for the proposed grant period are sharply focused on the degradation of HMG-CoA reductase which is a major mechanism regulating the level of reductase in the cell. This work promises fundamental insights into a novel problem in cell biochemistry, protein degradation in the endoplasmic reticulum, for which we have an excellent system for study. We will extend and refine our analysis of mutants in the membrane domain of HMG-CoA reductase in order to more precisely define the subregions or residues essential for degradation. For these experiments, we will continue to use as a model, the fusion protein, HMGal, which contains the membrane domain of HMG-CoA reductase fused to beta-galactosidase and responds to regulatory signals like native reductase. We will initiate an analysis of the biochemical components involved in the regulated degradation of reductase with particular attention to the protease(s). We plan to purify ALLN sensitive, ER protease(s). We have shown previously that the protease inhibitor peptide ALLN inhibits reductase degradation in vivo. We now have ALLN resistant cells with accelerated reductase degradation and elevated protease levels and these cells will be used as starting material for purification. We will characterize mutants of CHO cells which are altered in their ability to degrade reductase. We have successfully used a selection based on the fusion protein, HMhyg, which consists of the membrane domain of reductase fused to hygromycin phosphotransferase which confers hygromycin resistance upon cells in the absence of mevalonate but not in the presence. We have isolated two mutants which exhibit unregulated, rapid degradation of HMhyg, HMGal and HMG-CoA reductase. The genetic characterization of these mutants will lead to a biochemical understanding of the degradation process. We will also exploit a newly developed in vitro system for studying the degradation of reductase which uses permeabilized cells and exhibits physiologically relevant in vitro degradation. This system will be critical in directly defining the cellular location and biochemical components involved in the process.